Molecular screening of Entamoeba spp. (E. histolytica, E. dispar, E. coli, and E. hartmanni) and Giardia intestinalis using PCR and sequencing

A wide range of intestinal protozoan parasites inhabit the human gut. To establish a more comprehensive molecular screening, we designed PCR-sequencing screening methods for Entamoeba spp., including commensal species, and Giardia intestinalis, and performed such methods using 174 stool samples collected from Kenyan children. The prevalences of the target species were as follows: E. histolytica (2/174, 1.1%), E. dispar (20/174, 11.5%), E. coli (107/174, 61.5%), E. hartmanni (77/174, 44.3%), and G. intestinalis (54/174, 31.0%). PCR amplicons specific to G. intestinalis was differentiated to assemblages A (8/174, 4.6%) and B (46/174, 26.4%). PCR specificity for Entamoeba spp. was quite high, except for some cross-reactions between E. hartmanni detection primers and G. intestinalis, although the false-positive amplicons were discernible by the band size. The 18S rRNA PCR primers that was designed by Monis et al. in 1999 for G. intestinalis, have specificity issue, therefore amplicon sequencing was essential not only to determine assemblage classifications but also to confirm the positive results by eliminating potential non-specific reactions. The detection sensitivity of both the Entamoeba universal PCR and the G. intestinalis PCR was more than 100 copies of the target loci, which is sufficient for detecting a single trophozoite or cyst of both species.


Background
In this molecular screening of intestinal protozoan parasites, we initially targeted three main pathogens: Cryptosporidium spp., Entamoeba histolytica , and Giardia intestinalis .However, we did not detect any Cryptosporidium (data not shown).Using self-designed universal primer sets for Entamoeba screening, we found the presences of various non-pathogenic Entamoeba spp., with only two positive results for E. histolytica , while the detection rate of G. intestinalis was relatively high.The universal polymerase chain reaction (PCR) for Entamoeba can detect various Entamoeba spp.theoretically, including E. histolytica, E. coli, E. hartmanni, E. moshkovskii, E. bangladeshi, E. polecki, E. muris, E. nuttalli , and E. invadens .The ability of this method to detect almost all species of Entamoeba is an advantage over previous screening methods.In developing tropical areas, a wide range of intestinal protozoan parasites inhabit the human gut.The combination of this universal PCR for Entamoeba spp.and the conventional Giardia screening PCR could be a convenient tool for molecular screening such commensal species and mild pathogens.

Experimental design
In this study, we present a comprehensive molecular screening for Entamoeba spp.[1] and G. intestinalis [2] using stool samples collected from Kenyan children during a molecular epidemiologic investigation.
(1) Stool sample collection and DNA extraction Stool samples were collected from 174 schoolchildren in Kibera, Nairobi, Kenya in November 2017.Briefly, 0.2 g of stool sample was transferred into 1.5 mL screw-capped tubes containing 0.6 mL of DNAzol R ○ reagent (Molecular Research Center, Inc., Cincinnati, OH, USA).The collected samples were stored at 20 to 30 °C at the field site and at 4 °C in the laboratory for further analysis.
Total DNA was extracted from all DNAzol R ○ samples according to the manufacturer's instructions with a few modifications [1] .
Briefly, the samples were freeze-thawed twice, digested using proteinase K (final concentration 0.4 mg/mL, Wako Pure Chemical Industries, Osaka, Japan) at 55 °C overnight, and then precipitated using the standard ethanol precipitation protocol of the DNAzol R ○ reagent.The final DNA precipitate was resuspended in 80 μL of 10 mM Tris-HCl (pH 8.0) containing 1.0 mM ethylenediaminetetraacetic acid and stored at -20 °C until use.This study was approved by the Scientific and Ethics Review Unit of Kenya Medical Research Institute, Nairobi, Kenya (KEMRI/RES/7/3/1), and Kanazawa University, Japan (2379-1).Informed consent and assent were obtained from the study participants (children) and/or their parents or guardians.(2) Entamoeba spp.PCR screening For the molecular screening of Entamoeba spp.including E. histolytica, E. dispar, E. coli , and E. hartmanni , a set of universal nested PCR was performed to evaluate whether any Entamoeba spp. was present.The positive samples were further evaluated using Entamoeba species-specific PCR ( Fig. 1 A, Table 1 ).
For the universal Entamoeba spp.screening, a region of the 18S rRNA gene was amplified using nested PCR with the primers Amo-F1(TN21) and Amo-R1(TN14) in the first round and Amo-F2(MA115) and Amo-R2 (SA12) in the second round ( Table 1 ) [1] .It is worth mentioning that these universal primers were also 100% matched to the target genomic DNA region of other Entamoeba species, such as E. moshkovskii (KP722601), E. bangladeshi (KR025412), E. polecki (LC230018), E. muris (AB445018), E. nuttalli (AB282657), and E. invadens (KR025413).PCR amplification was performed in a 10 μl reaction mixture containing 1 × LA Taq R ○ PCR buffer, 0.5 U LA Taq R ○ polymerase (TaKaRa Bio Inc., Shiga, Japan), approximately 100 ng of the extracted DNA (1 μL) as the template, 0.4 μM of the forward and reverse primers, and 0.5 mM of deoxynucleotide triphosphates (dNTPs) with 0.1% dimethyl sulfoxide.The second round of PCR was performed under the same conditions as the first round PCR, except that 1.0 μL of the amplicon from the first round of PCR was used as the template.The cycling parameters for the first and second rounds of PCR are listed in Table 2 .
For Entamoeba species-specific PCR, specific multiplex primer sets (Eh-L, Eh-R, Ed-L, and Ed-R) were used for E. histolytica (product size, 427 bp) and E. dispar (194 bp), which were designed by Evangelopoulos et al. [3] , while all other species-specific primers were self-designed [1] .The species-specific primers used are listed in Table 1 .The Amo-R3 primer sequence was 100% matched to the target regions in E. histolytica (AB282660), E. dispar (AB282661), E. coli (AB444953), E. hartmanni (KX618191), E. moshkovskii (KP722601), E. bangladeshi (KR025412), E. polecki (LC230018), E. muris (AB445018), and E. nuttalli (AB282657).This means that the specificities of the PCRs for E. coli and E. hartmanni were ensured by the primers Ec-F(MA113) and Er-F(MA67), respectively.Same PCR conditions as those used in the second round of universal PCR were performed ( Table 2 ).All PCR cycles were conducted using the 2720 Thermal Cycler (Thermo Fisher Scientific Inc., MA, USA).Although all PCR specificities were confirmed in a previous study [1] , we observed slightly higher bands than the true positive one on the gel image of E. hartmanni-specific PCR in this study.For sequencing analyses, those slightly higher-seized PCR products were 5 ′ -565 CACTATTGGAGCTGGAATTAC 546 -3 ′ * Position numbers are based on the E. histolytica reference sequence (AB282660).
excised from the agarose gel after electrophoresis and then purified using the FastGene R ○ Gel/PCR Extraction Kit (Nippon Genetics, Tokyo, Japan).The purified amplicons were sequenced using amplification primers and/or appropriate sequencing primers on the 3130 Genetic Analyzer (Thermo Fisher Scientific Inc., MA, USA) using the BigDye R ○ Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific Inc.).All confirmed DNA sequences were registered in a DNA database [DNA Data Bank of Japan (DDBJ), European Molecular Biology Laboratory (EMBL), GenBank], with accession numbers LC732502-LC732508.The slightly larger PCR products amplified using E. hartmanni -specific primers were all identified as products resulting from cross-reaction with giardial DNA.(3) Giardia intestinalis PCR screening and sequencing The semi-nested and species-specific molecular screening of Giardia intestinalis were conducted, and genotyping was confirmed using the DNA sequences of the positive amplicons ( Fig. 1 B, Table 3 ).For the G. intestinalis -specific primers, the first PCR primer set was designed based on a set of primers G18S2 (5 ′ -TCCGGTYGATT * CTGCC-3 ′ ) and G18S3 (5 ′ -CTGGAATTACCGCGGCTGCT-3 ′ ), which was originally designed by Monis et al. [4] .However, the "T * " on the primer did not match the conserved region of the current giardial sequence alignment and an additional "G " was further conserved at the 3 ′ -end.Therefore, in this study, we modified the G18S2 primer to Gia-F(KN63) (5 ′ -TCCGGTYGATCCTGCCG-3 ′ ).For the second round of G. intestinalis -specific PCR, the inner region of the first PCR product was targeted, and the primer set Gia-F(KN63) (5 ′ -TCCGGTYGATCCTGCCG-3 ′ ) and Gia-R2 (MA64) (5 ′ -CCCGTCGCTGCCTCGCG-3 ′ ) was designed and used.PCR screening was performed in a 10 μl reaction mixture containing 1 × GC buffer I, 0.4 μM of each primer, 0.5 mM of each deoxynucleotide triphosphate (dNTP), and 0.5 U of TaKaRa LA Taq R ○ with GC Buffer (TaKaRa Bio Inc.), with 5% dimethyl sulfoxide as an additive.The cycling parameters for the first and second PCRs are listed in Table 2 .
To confirm the identity of G. intestinalis and the assemblage classification of G. intestinalis , all DNA sequences of the second PCR amplicons were determined directly using Gia-F (KN63) and/or Gia-R2 (MA64) primers.Purification and DNA sequencing methods were the same as those described above for Entamoeba species.For the assemblage classification of G. intestinalis , the alignment of amplicon sequences and Bayesian Inference analyses were conducted using Geneious 10.2.3 (Biomatters Ltd., Auckland, New Zealand).All confirmed 18S rRNA partial sequences of G. intestinalis were registered in the DNA database (DDBJ/EMBL/GenBank) with accession numbers LC732448-LC732501.(4) PCR sensitivity test for the universal PCR targeting 18S rRNA gene locus for Entamoeba spp.and G. intestinalis To conduct sensitivity tests, we constructed recombinant plasmids of pMD20-T using amplicons from the first PCR targeting the 18S rRNA gene locus.Specifically, we used a 1881-bp amplicon by primers Amo-F1(TN21) and Amo-R1(TN14) to test for E. histolytica , and a 464-bp amplicon by primers Gia-F (KN63) and G18S3 (KN62) to test for G. intestinalis .The TaKaRa TA-Mighty Cloning Kit (Takara Bio Inc.) was used for cloning following the manufacturer's instructions.The resulting recombinant plasmids (pMD20-T with those amplicons mentioned above) were transformed into Escherichia coli DH5- and screened on Luria Broth (LB) agar plates containing 100 mg/L Ampicillin.Positive colonies were cultured overnight in LB liquid medium with 100 mg/L Ampicillin and then purified using the QIAGEN R ○ plasmid mini kit (Qiagen K.K., Tokyo, Japan) according to the manufacturer's protocol.
The full-length insertion DNA was sequenced using M13 primer RV and M13 primer M4, and the amplicons were confirmed.The concentration of the plasmids was measured as double-stranded (ds) DNA using the OD 260 nm of μDrop TM (Thermo Fisher Scientific K.K., Tokyo, Japan), and the copy numbers were calculated based on their molecular weights.The detection limits of the universal PCR screenings for Entamoeba spp.and G. intestinalis were evaluated using dilution series of the templates.

Data measurement and analysis
PCR products of the universal PCR screening for Entamoeba spp.were electrophoresed on 2.0% agarose S gels (Nippon Gene, Toyama, Japan), though other PCR products were all electrophoresed on 1.0% agarose gels and visualized using ethidium bromide on a Gel Doc TM EZ Imaging System (Bio-Rad Laboratories, Tokyo, Japan).All electrophoresed images are shown in the Supplementary Figs.1-4, and only representative images are shown in Fig. 2 ).
(A) Entamoeba spp.second universal PCR .M, 1 kb DNA Ladder; N, negative control; P, positive control ( E. histolytica : 1,191bp); red colored sample, positive; white colored sample, negative; * asterisk shows that the faint band samples, which were verified via sequencing.The faint band DNA sequence of no.41 sample was unreadable.(B) Specific multiplex PCR for Entamoeba histolytica (Eh) and E. dispar (Ed) .M, 100 bp DNA Ladder; N, negative control; P, positive control; blue colored sample, Eh positive: 427 bp; red colored sample, Ed positive: 194 bp; white colored sample, negative.(C) Specific PCRs for Entamoeba coli (Ec) and E. hartmanni (Er) .M, 100 bp DNA Ladder; N, negative control; P, positive control; Red colored sample, Ec positive: 349 bp; Blue colored sample, Er positive: 419 bp; white colored sample, negative.* In Er screening lanes, slightly higher sized bands than the positive band of Er were observed, and thus checked via sequencing.Those were all determined as the cross-reacted products of G. intestinalis (sample 12: Gi cross-reaction LC732502).(D) Specific PCR for Giardia intestinalis (Gi) and sequencing for assemblages A and B .M, 100 bp DNA Ladder; N, negative control; P, positive control: 382 bp.The assemblage genotyping was confirmed using amplicon sequencing.Blue colored sample, Gi assemblage A; red colored sample, Gi assemblage B; yellow colored sample, sequenced but not detected Gi; white colored sample, no sequence data available.
(1) Entamoeba spp.second universal PCR ( Fig. 2 A) The positive bands of Entamoeba spp.varied in size: E. histolytica (1,192 bp), E. dispar (1,201 bp), E. hartmanni (1,205 bp), and E. coli (1,310 bp).The band intensity was generally strong; therefore, positive identification was relatively clear.Faint bands that rarely appeared were considered as non-specific reaction results because sequencing did not identify any cases of Entamoeba species.(2) Multiplex PCR specific for E. histolytica and E. dispar ( Fig. 2 B) Gel imaging revealed various non-specific amplicon bands; however, the size of non-specific bands was generally more than 500 bp, and thus the positive bands of E. histolytica (427 bp) and E. dispar (194 bp) were distinguishable.In previous sequencing assessments [1] , no cases of cross-reactions were observed in those amplicon bands derived from other organisms (data not shown).(3) E. coli-and E. hartmanni-specific PCR ( Fig. 2 C) Gel imaging revealed various non-specific amplicon bands; however, the size of such cross-reacted bands was more than 500 bp.Although problematic cross-reacted bands were not observed for the E. coli positive band (349 bp), slightly higher-sized bands than the positive band (419 bp) of E. hartmanni appeared.These higher-sized bands were all identified as cross-reacted products with G. intestinalis .For example, the band of sample 12, which was identified as the cross-reacted partial 18S rRNA gene fragment of G. intestinalis (LC732502).All other images of these cross reactions are shown in Supplementary Fig. 2.Even slightly, the band size was different from that of the true positive one; thus, this cross-reaction was distinguishable from the positive control.To confirm the presence of Giardia , all cross-reacted samples should be eliminated by sequence evaluation.For example, sample 1 was the assemblage B of G. intestinalis , sample 7 was assemblage A of G. intestinalis , and samples 2, 6, 8, and 10 were unreadable by sequencing.Non-specific band amplification seemed to be suppressed by the positive band amplification of the true Giardia template, probably due to the competitive use of nucleotide materials; however, sequencing is required to confirm the result.Assemblages of G. intestinalis were determined using those G. intestinalis positive amplicon reads by the alignment analysis (Supplementary Fig. 5).( 5) The sensitivity of the universal screening PCRs targeting the 18S rRNA gene locus for Entamoeba spp.and G. intestinalis Universal PCR screening for E. histolytica and G. intestinalis were both able to detect more than 100 copies of tested locus of DNA templates ( Fig. 3 ).
The 18S rRNA gene locus of E. histolytica is known to contain at least 200 copies in the cytoplasm as a plasmid [5] , and therefore, this screening PCR might retain sufficient sensitivity to detect a single trophozoite or cyst of E. histolytica .
G. intestinalis possesses five major chromosomes that are capped at both ends by telomeric tandem repeats containing 18S rRNA gene locus [6] .Even with a minimum of two repeats, each genome of G. intestinalis could possess at least 20 copies of the 18S rRNA locus.Additionally, due to the tetraploid nature of giardial karyotype, each trophozoite maintains at least 80 copies of the 18S rRNA locus.Considering these factors, the screening PCR's sensitivity might be sufficient for detecting a single trophozoite or cyst of G. intestinalis .

Conclusion
A comprehensive molecular screening method was performed using 174 stool samples collected from Kenyan children.The prevalence of Entamoeba spp.and G. intestinalis were as follows: E. histolytica (2/174, 1.1%), E. dispar (20/174, 11.5%), E. coli (107/174, 61.5%), E. hartmanni (77/174, 44.3%), and G. intestinalis (54/174, 31.0%).Sequencing of PCR amplicons specific to G. intestinalis (382 bp) could differentially identified assemblages A (8/174, 4.6%) and B (46/174, 26.4%).The specificity of all PCR cycles for Entamoeba spp. was quite high, except for some cross-reactions of E. hartmanni detection primers with G. intestinalis, although the false-positive amplicons were discernible by the band size.In G. intestinalis screening, amplicon sequencing is generally required not only to determine assemblage classifications but also to confirm positive results by eliminating potential non-specific reactions.The detection sensitivity of both the Entamoeba universal PCR and the G. intestinalis PCR was more than 100 copies of the target loci, which is sufficient for detecting a single trophozoite or cyst of both species.

Table 1
Primers targeting the 18S rRNA gene locus of Entamoeba spp.

Table 3
Primers targeting the 18S rRNA gene locus of Giardia intestinalis .